Liquid chromatography/mass spectrometry (LC/MS) is a widely used technique for the global identification and quantitation of proteins and peptides in complex biological samples. In this technique, liquid chromatography is used in-line with a mass spectrometer to chromatographically separate components prior to mass detection, in order to reduce the number of components presented to the mass spectrometer at a given time.
Liquid chromatography is an analytical chromatographic technique that is useful for separating components, typically ions or molecules, that are dissolved in a solvent. In this technique, the components (e.g., analytes) are first dissolved in a solvent and then are forced to flow through a chromatographic column that can range from a few centimeters to several meters. The column is packed with a solid phase chromatographic material that is matched to the solvents in use and binds the analytes via adsorption. An additional, different solvent is then mixed into the flow in increasing concentrations (such as by a smooth gradient increases, or step-wise increases, for example). Each compound in the analyte releases from the solid phase at a specific concentration of the additional solvent and then flows off of the column resulting in a serial separation of the compounds contained in the analyte. A variety of detectors for identifying the presence of compounds in the effluent have been developed over the past thirty years based on a variety of different sensing principles. Typically, signal intensity from a chromatographic detector can be plotted as a function of elution time (a chromatogram) and peaks are used to identify the components. Other techniques, such as characteristic retention time in a chromatographic column, may also be applied to identify the components. A mass spectrometer in this application functions as a very sensitive, multiplexed detector that can detect the presence of multiple compounds simultaneously and can differentiate between the compounds detected.
The evolution of mass spectrometry has been marked by an ever-increasing demand for improved sensitivity, resolution and mass accuracy and a wide variety of different techniques have been used to obtain them. However, at one level, the basic components of all mass spectrometers are essentially the same. These components may be best understood by tracing the ion's path through them. First, an ion source converts the analyte from the liquid (or solid) phase into the gas phase and places a charge on the molecules of the analyte. A common example of an ion source in an LC/MS system is electrospray ionization where the liquid phase input is sprayed into a chamber through a charged needle. Charge is deposited on the surface of the spray droplets and is transferred to the molecules of the analyte during the desolvation process where the solvents are evaporated off. Next, a mass analyzer differentiates the ions according to their mass-to-charge (m/z) ratio. Then, a detector measures the ion beam current to yield an m/z spectrum, where the peaks in the m/z spectrum may be used to differentiate and identify the input components.
A mass spectrometer produces a mass spectrum (m/z versus intensity) integrated over a finite interval of time. In the direct coupling of a liquid chromatography (LC) apparatus with a mass spectrometer (MS), each of these spectra represent an integrated view of the components coming off of the LC column over that interval. The mass spectrometer is typically set to gather a spectrum for a fixed repeating interval (e.g., over a period of five seconds, or some other preset interval). A single spectrum is commonly referred to as a scan and the repetition interval is referred to as the scan rate. The result is a set of ordered, two-dimensional spectra that can be treated as a single, three-dimensional data set, where the X-axis of the three-dimensional space represents elution time, the Y-axis represents m/z values and the Z-axis represents intensity. When using high resolution instruments under conditions where there is a large number of data points in each spectrum or scan, running the instruments at a high scan rate can result in output data sets which are very large and unwieldy (e.g., on the order of one gigabyte and greater).
An important aspect of analyzing LC/MS datasets involves peak detection to identify the ion current associated with each eluting component. As mentioned, peak detection is traditionally performed in a single dimension at a time, either in the chromatographic dimension (a chromatogram), where the intensity at each time point is the sum of the intensities over a given m/z range, or in the mass-to-charge (m/z) dimension (an m/z spectrum), where the intensity at each mass point represents the sum of intensities over a given time range. Peaks identified in this fashion may contain significant quantities of contaminating signal (e.g., from noise or adjacent compounds) and hence this approach to identification typically requires significant knowledge as to the behavior of the components/analytes being studied, to increase the probability that all of the ion current associated with a particular peak is considered when analyzing the same.
Quantitation generally refers to the processing step or steps involved in determining an amount or quantity of molecule rather than identifying a particular type or types of molecules. Quantitation may be performed, at least in part, by integrating the total ion current associated with a particular peak representing the ion of interest. When peak detection is performed in only one dimension, either in elution time using ion chromatograms or in m/z using spectra, the resulting contamination and/or missing ion current can result in significant inaccuracy of the quantitation results.
While two dimensional peak assessment has been attempted with regard to nuclear magnetic resonance (NMR) data analysis, such techniques have not been successful with regard to mass spectrometry/elution time data, as they have not performed well due to the localized nature of noise observed in such datasets, (e.g., LC/MS datasets). As a result, most peak detection methods for LC/MS and other mass spectrometry/elution time datasets continue to be performed in one dimension, either in the m/z (spectral) or elution time (chromatograph) dimensions.
Thus, there is a need to provide a method for peak detection and quantitation of large datasets such as LC/MS datasets and other mass spectrometry/elution time datasets in the elution time and m/z dimensions simultaneously. Such methods may be applicable for the analysis of proteins as well as other classes of molecules sharing similar characteristics.